Chapter 4: An Inducible, Isogenic Cancer Cell Further Validates
5.2 Experimental Protocols
5.2.4 X-Ray Structure Determination
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8.32-8.44 (m, 5H); 8.17 (m, 2H); 8.08 (m, 1H); 7.91 (t, J = 7.0 Hz, 1H); 7.86 (t, J = 8.0 Hz, 1H); 7.80 (m, 2H); 7.54 (t, J = 7.5 Hz, 1H); 7.41 (d, J = 8.0 Hz, 1H); 7.31 (d, J = 6.0 Hz, 1H); 7.05 (t, J = 7.0 Hz, 1H); 1.74 (s, 3H); 1.55 (m, 2H); 0.71-0.96 (m, 11H).
Crystals suitable for X-ray diffraction were obtained from vapor diffusion of diethyl ether into a concentrated solution of [Rh(chrysi)(phen)(DPE)]Cl2 dissolved in ethanol.
Crystals of [Rh(HDPA)2(chrysi)]Cl2 (one chrysi immine deprotonated) suitable for X-ray diffraction were obtained from vapor diffusion of diethyl ether into a concentrated solution of [Rh(HDPA)2(chrysi)]Cl2 dissolved in ethanol.
5.2.3.3 Enantiomeric Separation
1.5 mL of a 2 mM solution of [Rh(chrysi)(phen)(DPE)]2+ was injected, 30 L at a time, onto an Astec® CYCLOBOND® I 2000 Chiral HPLC Column that was heated to 40o C. An isocratic method of 50% acetonitrile, 50% 100 mM KPF6 was used to separate the two enantiomers. An automatic fraction collector was used to collect each peak separately. The resulting dilute solutions were loaded onto a SPE cartridge and rinsed with copious amount of 0.1% TFA(aq). The SPE cartridge was eluted with 10%
acetonitrile in 0.1% TFA(aq). The chloride salts were obtained from a Sephadex QAE anion exchange column equilibrated with 0.1M MgCl2. Circular dichroism spectra were taken on an Aviv 62DS spectropolaarimeter in a 1 mm path length cell.
5.2.4 X-Ray Structure Determination
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Mo K radiation ( = 0.71073 Å) for the structure of [Rh(HDPA)2(chrysi)]Cl2. The structure was solved by direct methods using SHELXS6 and refined against F2 on all data by full-matrix least squares with SHELXL-20137 using established refinement techniques.8 All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were included into the model at geometrically calculated positions and refined using a riding model. The isotropic displacement parameters of all hydrogen atoms were fixed to 1.2 times the U value of the atoms they are linked to (1.5 times for methyl groups).
[Rh(HDPA)2(chrysi)]Cl2 crystallizes in the monoclinic space group P21/n with one molecule in the asymmetric unit along with two molecules of methanol. One of the two chloride ions was disordered over two positions and refined with the help of similarity restraints on the displacement parameters. The occupancies of the two components refined to 0.930(4):0.070(4). The coordinates for the hydrogen atoms bound to N1, N4, N7, O1S, O1T, and O1W were located in the difference Fourier synthesis and refined semi-freely with the help of a distance restraint. The N-H distances were restrained to be 0.91(4) Å for N1, 0.88(4) Å for N4, N7 and 0.84(4) Å for all O-H bonds.
5.2.4.2 [Rh(chrysi)(phen)(DPE)]Cl2
Low-temperature diffraction data (-and -scans) were collected on a Bruker Kappa diffractometer coupled to an Apex II CCD detector with graphite-monochromated Mo K radiation ( = 0.71073 Å) for the structure of [Rh(chrysi)(phen)(DPE)]Cl2. The structure was solved by direct methods using SHELXS6 and refined against F2 on all data by full-matrix least squares with SHELXL-20137 using established refinement techniques.8 All non-hydrogen atoms were refined anisotropically. Unless otherwise noted, all hydrogen atoms were included into the model at geometrically calculated
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positions and refined using a riding model. The isotropic displacement parameters of all hydrogen atoms were fixed to 1.2 times the U value of the atoms they are linked to (1.5 times for methyl groups).
[Rh(chrysi)(phen)(DPE)]Cl2 crystallizes in the triclinic space group P ̅with one molecule in the asymmetric unit along with two chloride anions, two waters, and two molecules of ethanol. Each of the ethanol molecules were disordered over three positions. They were refined with the help of similarity restraints on the 1,2- and 1,3- distances and displacement parameters as well as rigid bond restraints for anisotropic displacement parameters. The occupancies of the two disordered ethanol molecules were freely refined to 0.509(3):0.261(3):0.229(3) and 0.453(3):0.295(3):0.252(3), respectively.
Additional residual electron density is located near each of the disordered ethanol molecules. However, the refinement of additional ethanol positions was unsuccessful, and the current model represents the best model which led to a stable refinement. The coordinates for the hydrogen atoms bound to N1, N2, O1W and O2W were located in the difference Fourier synthesis and refined semi-freely with the help of a distance restraint.
The N-H and O-H distances were restrained to be 0.88(4) Å and 0.84(4) Å, respectively.
The hydrogen atoms bound to oxygen in the ethanol molecules could not be found and were included at geometrically calculated positions and refined using a riding model.
5.2.4.3 [Rh(chrysi)(phen)(PPE)]Cl2
Low-temperature diffraction data (-and -scans) were collected on a Bruker Kappa diffractometer coupled to an Apex II CCD detector with graphite- monochromated Mo K radiation ( = 0.71073 Å) for the structure of [Rh(chrysi)(phen)(PPE)]Cl2. The structure was solved by direct methods using
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SHELXS6 and refined against F2 on all data by full-matrix least squares with SHELXL- 20138 using established refinement techniques.8 All non-hydrogen atoms were refined anisotropically. Unless otherwise noted, all hydrogen atoms were included into the model at geometrically calculated positions and refined using a riding model. The isotropic displacement parameters of all hydrogen atoms were fixed to 1.2 times the U value of the atoms they are linked to (1.5 times for methyl groups). All disordered atoms were refined with the help of similarity restraints on the 1,2-distances and displacement parameters as well as rigid bond restraints for anisotropic displacement parameters.
[Rh(chrysi)(phen)(PPE)]Cl2 crystallizes in the triclinic space group P ̅ with one molecule in the asymmetric unit along with two chloride anions and four molecules of methanol. One of the methanol molecules hydrogen-bonds to the ruthenium molecule and was not disordered. The three other methanol molecules were modeled as disordered. For the second methanol, only the methyl group was disordered over two positions with the occupancy of the two components refined to 0.841(7):0.159(7). The third methanol was completely disordered over two positions with occupancies of 0.792(6):0.208(6). The fourth methanol was disordered over three positions with occupancies 0.0615(3):0.234(3):0.151(3). The coordinates for the hydrogen atoms bound to N1, N2, and O1M were located in the difference Fourier synthesis and refined semi- freely with the help of a distance restraint. The N-H and O-H distances were restrained to be 0.88(4) Å and 0.84(4) Å, respectively. The hydrogen atoms for the remaining methanol positions could not be located and were included at geometrically calculated positions and refined using a riding model.
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5.2.4.4 [Rh(chrysi)(phen)(PPO)]Cl2
Low-temperature diffraction data (-and -scans) were collected on a Bruker three-circle diffractometer coupled to a Bruker Smart 1000 CCD detector with graphite monochromated Mo K radiation ( = 0.71073 Å) for the structure of compound ack013.
The structure was solved by direct methods using SHELXS6 and refined against F2 on all data by full-matrix least squares with SHELXL-20137 using established refinement techniques.8 All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were included into the model at geometrically calculated positions and refined using a riding model. The isotropic displacement parameters of all hydrogen atoms were fixed to 1.2 times the U value of the atoms they are linked to (1.5 times for methyl groups).
Compound ack013 crystallizes in the triclinic space group P ̅ with one molecule in the asymmetric unit along with two chloride anions, one molecule of diethyl ether, and one molecule of isopropyl alcohol. The coordinates for the hydrogen atoms bound to N1, N2, and O1T were located in the difference Fourier synthesis and refined semi-freely with the help of a distance restraint. The N-H and O-H distance were restrained to be 0.88(4) Å and 0.84(4) Å, respectively.